Compounds and their use for the treatment of alpha1-antitrypsin deficiency

ABSTRACT

The invention relates to oxopyrimidinyl-methyl-benzamide derivatives, for example in a pharmaceutically acceptable salt form or crystal form, pharmaceutical compositions comprising the derivatives, and their medical use, in particular for use in the treatment of α 1 -antitrypsin deficiency (A1AD or AATD).

CROSS-REFERENCE

This application is a continuation of U.S. application Ser. No.17/345,928, filed Jun. 11, 2021, which is the by-pass continuation ofInternational Application No. PCT/GB2019/053552, filed Dec. 13, 2019,which claims the benefit of GB Application No. 1820450.3, filed Dec. 14,2018, each of which are incorporated herein by reference in theirentireties.

SUMMARY

The invention relates toN,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide and relatedcompounds, and their medical use.

α₁-Antitrypsin (A1AT) is a member of the serpin superfamily produced bythe liver and secreted into the blood. It inhibits a variety of serineproteases, especially neutrophil elastase. When blood levels of A1AT arelow, excessive neutrophil elastase activity degrades lung tissueresulting in respiratory complications such as chronic obstructivepulmonary disease (COPD).

The reference range of A1AT in blood is 0.9-2.3 g/L. Levels lower thanthis are typical of α₁-antitrypsin deficiency (A1AD or AATD), a geneticdisorder caused by mutations in the SERPINA1 gene, coding for A1AT. TheZ mutation, the most common cause of AATD, is the substitution ofglutamate to lysine at position 366 of A1AT (UniProtKB-P01009(A1AT_HUMAN)), corresponding to position 342 in the mature protein (ZA1AT). The Z mutation affects the folding of A1AT resulting in only asmall fraction acquiring the native/active state. The remainder iseither cleared as misfolded protein or accumulates in the liver asstable polymers. As a consequence of the misfolding, homozygous carriersof the Z mutation (ZZ) have plasma levels of A1AT that are 10-15% ofnormal, predisposing carriers to COPD. Accumulation of Z A1AT polymersin liver cells predisposes carriers to cirrhosis, liver cancer and otherliver pathologies.

The current treatment for the lung manifestation of AATD involvesaugmentation therapy using A1AT concentrates prepared from the plasma ofblood donors. The US FDA has approved the use of four A1AT products:Prolastin, Zemaira, Glassia, and Aralast. Dosing is via once weeklyintravenous infusion. Augmentation therapy has been demonstrated to slowprogression of COPD. The liver manifestations of AATD (e.g. cirrhosisand cancer) are treated with steroids and liver transplantation.Investigational approaches to improved treatment of the livermanifestations include inhibition of Z A1AT polymerisation and increasedclearance of polymers through the activation of autophagy.Investigational approaches to improved treatment of both the lung andthe liver manifestations are directed towards improvement of Z A1ATfolding and secretion.

Elliott et al (Protein Science, 2000, 9, 1274-1281) have described anX-ray crystal structure of AAT and identified five cavities that arepotential targets for rational drug design to develop agents that willaffect Z A1AT polymerisation.

Parfrey et al (J. Biol. Chem., 2003, 278, 35, 33060-33066) have furtherdefined a single cavity that is a potential target for rational drugdesign to develop agents that will affect Z A1AT polymerisation.

Knaupp et al (J. Mol. Biol., 2010, 396, 375-383) have shown that bis-ANS(4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonate) is able to bind to ZA1AT, but not to wild-type, A1AT (M), with 1:1 stoichiometry and a K_(d)of 700 nM.

Chang et al (J. Cell. Mol. Med., 2009, 13, 8B, 2304-2316) have reporteda series of peptides, including Ac-TTAI-NH₂, that inhibit Z A1ATpolymerization.

Burrows et al (Proc. Nat. Acad. Sci., 2000, 97, 4, 1796-1801) have shownthat a series of non-selective chaperones, including 4-phenylbutyricacid, glycerol and trimethylamine oxide, are able to increase Z A1ATlevels in cell supernatants and mouse models.

Bouchecareilh et al (Journal of Biological Chemistry, 2012, 287, 45,38265-38278) describe the use of histone deacetylase inhibitors, inparticular SAHA (suberoylanilide hydroxamic acid) to increase thesecretion of both wild-type (M) and Z A1AT from cells.

Berthelier et al (PLOS ONE, May 11, 2015) have demonstrated thatS-(4-nitrobenzyl)-6-thioguanosine is able to prevent Z A1ATpolymerisation in vitro.

Mallya et al (J. Med. Chem., 2007, 50, 22, 5357-5363) describe a seriesof phenols, such asN-(4-hydroxy-3,5-dimethylphenyl)-2,5-dimethylthiophene-3-sulfonamide,able to block polymerisation of Z A1AT in vitro.

Huntington (XIIIth International Symposium on Proteinases, Inhibitorsand Biological Control, 23 Sep. 2012, and 7^(th) International Symposiumon Serpin Biology, Structure and Function, 1 Apr. 2014) discussed acavity from an X-ray crystal structure of Z A1AT that is a potentialtarget for rational drug design to develop agents that will affect ZA1AT polymerisation.

U.S. Pat. No. 8,436,013B2 discloses a wide variety of structures able toincrease secretion of Z A1AT from cells in the micromolar range.

Compounds with CAS registry numbers 1797054-78-4 and 1219580-65-0 arelisted in the Aurora Building Blocks catalogue.

According to one aspect of the present invention, there is providedN,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide:

We have found thatN,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide is shownsurprisingly to be highly effective at increasing levels of correctlyfolded, and hence active, Z A1AT, whilst having no effect on thesecretion of wild type (M) A1AT or of the Siiyama variant of A1AT.

According to another aspect of the present invention, there is providedN-methyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide:

We have found thatN,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide is shownsurprisingly to be highly effective at increasing levels of correctlyfolded, and hence active, Z A1AT, whilst having no effect on thesecretion of wild type (M) A1AT or of the Siiyama variant of A1AT.

According to a further aspect of the present invention, there isprovidedN,N-bis(methyl-d₃)-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide:

We have found thatN,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide is shownsurprisingly to be highly effective at increasing levels of correctlyfolded, and hence active, Z A1AT, whilst having no effect on thesecretion of wild type (M) A1AT or of the Siiyama variant of A1AT.

The compound of the invention may be in a pharmaceutically acceptablesalt form or crystal form.

The term “pharmaceutically acceptable salt” refers to a pharmaceuticallyacceptable mono organic or inorganic salt of the compound of theinvention. This may include addition salts of inorganic acids such ashydrochloride, hydrobromide, hydroiodide, sulphate, phosphate,diphosphate and nitrate or of organic acids such as acetate, maleate,fumarate, tartrate, succinate, citrate, lactate, methanesulphonate,p-toluenesulphonate, palmoate and stearate. Exemplary salts also includeoxalate, chloride, bromide, iodide, bisulphate, acid phosphate,isonicotinate, salicylate, acid citrate, oleate, tannate, pantothenate,bitartrate, ascorbate, gentisinate, gluconate, glucuronate, saccharate,formate, benzoate, glutamate, ethanesulfonate, and benzenesulfonatesalts. For other examples of pharmaceutically acceptable salts,reference can be made to Gould (1986, Int J Pharm 33: 201-217).

According to a further aspect of the invention, there is a provided apharmaceutical composition comprising the compound of the invention asdescribed herein and a pharmaceutically or therapeutically acceptableexcipient or carrier.

The term “pharmaceutically or therapeutically acceptable excipient orcarrier” refers to a solid or liquid filler, diluent or encapsulatingsubstance which does not interfere with the effectiveness or thebiological activity of the active ingredients and which is not toxic tothe host, which may be either humans or animals, to which it isadministered. Depending upon the particular route of administration, avariety of pharmaceutically acceptable carriers such as those well knownin the art may be used. Non-limiting examples include sugars, starches,cellulose and its derivatives, malt, gelatin, talc, calcium sulfate,vegetable oils, synthetic oils, polyols, alginic acid, phosphatebuffered solutions, emulsifiers, isotonic saline, and pyrogen-freewater.

All suitable modes of administration are contemplated according to theinvention. For example, administration of the medicament may be viaoral, subcutaneous, direct intravenous, slow intravenous infusion,continuous intravenous infusion, intravenous or epidural patientcontrolled analgesia (PCA and PCEA), intramuscular, intrathecal,epidural, intracistemal, intraperitoneal, transdermal, topical,transmucosal, buccal, sublingual, transmucosal, inhalation, intranasal,intra-atricular, intranasal, rectal or ocular routes. The medicament maybe formulated in discrete dosage units and can be prepared by any of themethods well known in the art of pharmacy.

All suitable pharmaceutical dosage forms are contemplated.Administration of the medicament may for example be in the form of oralsolutions and suspensions, tablets, capsules, lozenges, effervescenttablets, transmucosal films, suppositories, buccal products, oralmucoretentive products, topical creams, ointments, gels, films andpatches, transdermal patches, abuse deterrent and abuse resistantformulations, sterile solutions suspensions and depots for parenteraluse, and the like, administered as immediate release, sustained release,delayed release, controlled release, extended release and the like.

Another aspect of the invention is the use of the compound of theinvention as defined herein in the manufacture of a medicament for thetreatment of a disease or disorder.

A further aspect of the invention is the compound of the invention foruse as an inducer of Z A1AT secretion.

Further provided is the compound of the invention as defined herein foruse in the treatment of a disease or disorder.

The invention also encompasses a method of treating a disease ordisorder, comprising the step of administering the compound or thepharmaceutical composition of the invention as defined herein to apatient in need of same.

The invention further encompasses the use of a compound of the inventionas an inducer of Z A1AT secretion. The use may be in the treatment of adisease or disorder. Additionally or alternatively, the use may be invitro, for example in an in vitro assay.

A disease or disorder suitable for treatment according to the relevantaspects of the invention is one which is characterised by low plasmalevels of A1AT, for example AATD.

The use of a numerical range in this description is intendedunambiguously to include within the scope of the invention allindividual integers within the range and all the combinations of upperand lower limit numbers within the broadest scope of the given range.

As used herein, the term “comprising” is to be read as meaning bothcomprising and consisting of Consequently, where the invention relatesto a “pharmaceutical composition comprising as active ingredient” acompound, this terminology is intended to cover both compositions inwhich other active ingredients may be present and also compositionswhich consist only of one active ingredient as defined.

Unless otherwise defined, all the technical and scientific terms usedhere have the same meaning as that usually understood by an ordinaryspecialist in the field to which this invention belongs. Similarly, allthe publications, patent applications, all the patents and all otherreferences mentioned here are incorporated by way of reference in theirentirety (where legally permissible).

Particular non-limiting examples of the present invention will now bedescribed with reference to the following drawings, in which:

FIG. 1 is a graph showing the dose dependent effect ofN,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide in an in vitroA1AT cell secretion assay using HEK-EBNA cells containing the Z A1ATplasmid. Vehicle and 10 μM SAHA were tested on each plate as controls.The x-axis shows various treatments of the cells: vehicle, SAHA andincreasing concentrations ofN,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide, the y-axis isthe concentration of A1AT in the cell supernatant (in ng/ml);

FIG. 2 is a graph showing the effect ofN,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide at 10 μM in anin vitro A1AT cell secretion assay using HEK-EBNA cells containing the MA1AT plasmid. Vehicle and 10 μM SAHA were tested as controls. The x-axisshows various treatments of the cells: vehicle, SAHA andN,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide, the y-axis isthe concentration of A1AT in the cell supernatant (in ng/ml);

FIG. 3 is a graph showing the effect ofN,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide at 1 and 10 μMin an in vitro A1AT cell secretion assay using HEK-EBNA cells containingthe Siiyama A1AT plasmid. Vehicle and 10 μM SAHA were tested ascontrols. The x-axis shows various treatments of the cells: vehicle,SAHA and two concentrations ofN,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide, the y-axis isthe concentration of A1AT in the cell supernatant (in ng/ml);

FIG. 4 is a graph showing the effect ofN,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide on Z A1ATlevels in mice expressing human Z A1AT (huZ mice). Mice were treatedwith vehicle, 5, 15 and 50 mg/kgN,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide twice a day byoral gavage for 14 consecutive days. Blood was taken on days −12, −7 and−5 and plasma prepared to determine circulating basal levels of human ZA1AT. Plasma samples collected on the last three days of the study (days12, 13 and 14) were used to determine the effect ofN,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide treatment oncirculating human Z A1AT levels compared to basal levels. The x-axis isthe treatment dose ofN,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide in mg/kg;they-axis is the mean percentage level of human Z A1AT compared tobaseline levels for each treatment group;

FIG. 5 is a graph showing the dose dependent effect ofN,N-(bis-methyl-d₃)-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide in anin vitro A1AT cell secretion assay using HEK-EBNA cells containing the ZA1AT plasmid. Vehicle and 10 μM SAHA were tested on each plate ascontrols. The x-axis shows various treatments of the cells: vehicle,SAHA and increasing concentrations ofN,N-(bis-methyl-d₃)-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide, they-axis is the concentration of A1AT in the cell supernatant (in ng/ml);

FIG. 6 is a graph showing the dose dependent effect ofN-methyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide in an in vitroA1AT cell secretion assay using HEK-EBNA cells containing the Z A1ATplasmid. Vehicle and 10 μM SAHA were tested on each plate as controls.The x-axis shows various treatments of the cells: vehicle, SAHA andincreasing concentrations ofN-methyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide, the y-axis is theconcentration of AAT in the cell supernatant (in ng/ml); and

FIGS. 7A and B show Western blots of Z A1AT after isolation andtreatment with trypsin. Z A1AT was isolated after treatment of one wildtype (15.5b) and three HuZ mice (22.1e, 24.1a, 24.1b) withN,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide at a doselevel of 200 mgKg by oral gavage bid for 10 days. Each Western blot islabelled with the identifier of the mouse. Gel lanes are labelledaccording to Table 4. Protein bands are labelled as “C”—Complex,“NA”—Native antitrypsin, and “CA”—Cleaved antitrypsin.

EXPERIMENTAL Example 1:N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide

N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide was preparedusing the following sequential synthesis procedures.

(a) Synthesis of Tert-Butyl 4-((6-oxopyrimidin-1(6H)-yl)methyl)benzoate

Pyrimidin-4(3H)-one (5 g, 32 mmol) and caesium carbonate (50.85 g, 156mmol) were stirred in dimethylformamide (50 ml) for 10 minutes at roomtemperature. Tert-butyl 4-(bromomethyl)benzoate (14.11 g, 52 mmol) wasadded and the reaction was stirred for 3 hours. The reaction was dilutedwith water and the resulting yellow precipitate collected by filtration.The crude product was purified by column chromatography on silica,eluting with ethyl acetate/hexane (30% to 33%) to give tert-butyl4-((6-oxopyrimidin-1(6H)-yl)methyl)benzoate. Tlc Rf 0.2 1:1 Ethylacetate/hexane.

(b) Synthesis of 4-((6-oxopyrimidin-1(6H)-yl)methyl)benzoic Acid

Tert-butyl 4-((6-oxopyrimidin-1(6H)-yl)methyl)benzoate (10 g, 35 mmol)was dissolved in dichloromethane (50 ml) and trifluoroacetic acid (70ml) was added slowly. The reaction was stirred for 3 hours at roomtemperature. The reaction was concentrated under reduced pressure andthe resulting oil stirred with diethyl ether (300 ml) for 20 minutes atroom temperature. The resultant solid was collected by filtration,washed with diethyl ether (2×30 ml) and dried in vacuo to give4-((6-oxopyrimidin-1(6H)-yl)methyl)benzoic acid.

(c) Synthesis ofN,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide

4-((6-oxopyrimidin-1(6H)-yl)methyl)benzoic acid (16 g, 69 mmol) andN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (18 g, 139mmol) were stirred in tetrahydrofuran (100 ml) for 10 minutes at 0° C.under nitrogen. The reaction was then allowed to warm to roomtemperature. Triethylamine (21.11 g, 208 mmol) and dimethylamine (2Msolution in tetrahydrofuran, 69 mmol) were added and the reaction wasstirred for 2 hours. The reaction was concentrated under reducedpressure and the residue columned on silica eluting with 4% methanol indichloromethane. Product containing fractions were concentrated to giveN,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide.

Tlc Rf 0.8 10% methanol in dichloromethane.

m/z: 257.96 (calc 258.12).

¹H NMR (400 MHz, d6 DMSO) δ 8.69 (1H, s), 7.94 (1H, d), 7.38 (4H, m),6.44 (1H, d), 5.13 (2H, s), 2.96 (3H, br s), 2.87 (3H, br s).

N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide could also beprepared by alkylation of pyrimidin-4(3H)-one with4-(bromomethyl)-N,N-dimethylbenzamide in a similar manner to step a).

Example 2: N-methyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide

N-methyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide was preparedsimilarly to N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamideusing methylamine instead of dimethylamine in step c.

m/z: 242.90 (calc 243.10).

1H NMR (400 MHz, d6 DMSO) δ 8.69 (1H, s), 8.42 (1H, br m), 7.94 (1H, d),7.79 (2H, d), 7.37 (2H, d), 6.43 (1H, d), 5.13 (2H, s), 2.77 (3H, s).

Example 3:N,N-(bis-methyl-d₃)-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide

N,N-(bis-methyl-d₃)-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide wasprepared similarly toN,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide usingd₆-dimethylamine instead of dimethylamine in step c.

Tlc Rf 0.8 10% methanol in dichloromethane.

¹H NMR (400 MHz, d6 DMSO) δ 8.69 (1H, s), 7.94 (1H, d), 7.38 (4H, m),6.44 (1H, d), 5.13 (2H, s).

Example 4: Activity of Compounds of the Invention in an A1AT CellSecretion Assay Using HEK-Z Cells

Methods

HEK-Z cells, a human embryonic kidney cell line stably transfected withthe human Z A1AT gene, were plated into 96 well plates (3.0×10⁵ cells/mlwith 200 μl of media/well) overnight at 37° C. in a humidifiedatmosphere containing 5% CO₂. Following incubation cells were washedwith 200 μl serum-free media three times and media was replaced withtreatments in quadruplicate using serum-free media containing eithervehicle, 10 μM suberanilohydroxamic acid (SAHA),N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide (atconcentrations of 10, 33, 100 and 333 nM),N,N-(bis-methyl-d3)-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide (atconcentrations of 10, 33, 100 and 333 nM) orN-methyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide (at concentrationsof 10, 33, 100 and 333 nM) for 48 h in a 37° C. incubator in a finalvolume of 200 μl. At the end of the incubation step the supernatantswere removed from the wells, centrifuged at 1000×g at 4° C. for 10 minand were assayed for human A1AT levels by ELISA (Human SerpinA1/α₁-antitrypsin duo set ELISA, R& D Systems, DY1268) permanufacturer's instructions.

Briefly, a 96 well plate was coated with human A1AT capture antibodyovernight at room temperature (1:180 dilution from stock, 100 μl finalvolume/well). The capture antibody was then removed and wells washedthree times with 300 μl wash buffer (0.05% Tween 20 in PBS) and then 200μl reagent diluent (25% Tween 20 in PBS) was incubated in each well for1 h at room temperature. Diluted samples, standards (125, 250, 500,1000, 2000, 4000 and 8000 pg/ml A1AT) or blanks were then added to eachwell in duplicate and the plates were covered with a plate sealer andleft at room temperature for 2 h. At the end of the sample incubationstep, samples were removed and all wells washed as previously and 100 μldetection antibody (1:180 dilution from stock) was added to each welland incubated for a further 2 h at room temperature. Followingincubation with detection antibody, supernatant was removed and wellswere washed as previously and 100 μl streptavidin-HRP solution (1:200dilution from stock) was added to each well for 20 min in the dark.After which, 50 μl stop solution (2M H₂SO₄) was added and opticaldensity (OD) of each well was read at 450 nm with 570 nm blanksubtracted from each well using a microplate reader. A 4 parameterlogistic curve was constructed using GraphPad Prism 7 and A1ATconcentrations were determined in each sample by interpolation from astandard curve and multiplying by the appropriate dilution factor.

Results

The amount of A1AT secreted from transfected HEK-EBNA cells into themedia was measured by ELISA. SAHA at 10 μM was used a positive controlfor all in vitro A1AT secretion experiments.

The data in FIG. 1 show thatN,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide stimulatessecretion of Z A1AT in a dose dependent manner as measured by ELISA.

The data in FIG. 5 show thatN,N-(bis-methyl-d₃)-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamidestimulates secretion of Z A1AT in a dose dependent manner as measured byELISA.

The data in FIG. 6 show thatN-methyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide stimulatessecretion of Z A1AT in a dose dependent manner as measured by ELISA.

Example 5: Activity of Compounds of the Invention in an A1AT CellSecretion Assay Using HEK-M Cells

Methods

HEK-M cells, a human embryonic kidney cell line stably transfected withthe human M A1AT gene, were plated into 96 well plates (3.0×10⁵ cells/mlwith 200 μl of media/well) overnight at 37° C. in a humidifiedatmosphere containing 5% CO₂. Following incubation cells were washedwith 200 μl serum-free media three times and media was replaced withserum-free media containing either vehicle, 10 μM suberanilohydroxamicacid (SAHA) or a compound of the invention (at 10 μM) in replicates ofsix for 48 h in a 37° C. incubator in a final volume of 200 μl. At theend of the incubation step the supernatants were removed from the wells,centrifuged at 1000×g at 4° C. for 10 min and were assayed for humanA1AT levels by ELISA (Human Serpin A1/α₁-antitrypsin duo set ELISA, R& DSystems, DY1268) per manufacturer's instructions.

Briefly, a 96 well plate was coated with human A1AT capture antibodyovernight at room temperature (1:180 dilution from stock, 100 μl finalvolume/well). The capture antibody was then removed and wells washedthree times with 300 μl wash buffer (0.05% Tween 20 in PBS) and then 200μl reagent diluent (25% Tween 20 in PBS) was incubated in each well for1 h at room temperature. Diluted samples, standards (125, 250, 500,1000, 2000, 4000 and 8000 pg/ml A1AT) or blanks were then added to eachwell in duplicate and the plates were covered with a plate sealer andleft at room temperature for 2 h. At the end of the sample incubationstep, samples were removed and all wells washed as previously and 100 μldetection antibody (1:180 dilution from stock) was added to each welland incubated for a further 2 h at room temperature. Followingincubation with detection antibody, supernatant was removed and wellswere washed as previously and 100 μl streptavidin-HRP solution (1:200dilution from stock) was added to each well for 20 min in the dark.After which, 50 μl stop solution (2M H₂SO₄) was added and opticaldensity (OD) of each well was read at 450 nm with 570 nm blanksubtracted from each well using a microplate reader. A 4 parameterlogistic curve was constructed using GraphPad Prism 7 and A1ATconcentrations were determined in each sample by interpolation from astandard curve and multiplying by the appropriate dilution factor.

Results

The amount of A1AT secreted from transfected HEK-EBNA cells into themedia was measured by ELISA. SAHA at 10 μM was used a positive controlfor all in vitro A1AT secretion experiments.

The data in FIG. 2 show thatN,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide does notstimulate secretion of M A1AT at 10 μM, as measured by ELISA. Incontrast, the positive control 10 μM SAHA stimulates an increase in MA1AT secretion.

Example 6: Activity of Compounds of the Invention in an A1AT CellSecretion Assay Using HEK-Siiyama Cells

The rare Siiyama mutation (Ser 53 to Phe, mature A1AT numbering) wasidentified in a Japanese male with AATD (Seyama et al J Biol Chem (1991)266:12627-32). Ser53 is one of the conserved serpin residues and isthought to be important for the organization of the internal core of theA1AT molecule. The change from an uncharged polar to a large nonpolaramino acid on the conserved backbone of the protein affects the foldingand intracellular processing of Siiyama A1AT.

Methods

HEK-Siiyama cells, a human embryonic kidney cell line stably transfectedwith the human Siiyama A1AT gene, were plated into 96 well plates(3.0×10⁵ cells/ml with 200 μl of media/well) overnight at 37° C. in ahumidified atmosphere containing 5% CO₂. Following incubation cells werewashed with 200 μl serum-free media three times and media was replacedwith serum-free media containing either vehicle, 10 μMsuberanilohydroxamic acid (SAHA) or a compound of the invention (at 1 or10 μM) in replicates of eight for 48 h in a 37° C. incubator in a finalvolume of 200 μl. At the end of the incubation step the supernatantswere removed from the wells, centrifuged at 1000×g at 4° C. for 10 minand were assayed for human A1AT levels by ELISA (Human SerpinA1/α₁-antitrypsin duo set ELISA, R& D Systems, DY1268) permanufacturer's instructions.

Briefly, a 96 well plate was coated with human A1AT capture antibodyovernight at room temperature (1:180 dilution from stock, 100 μl finalvolume/well). The capture antibody was then removed and wells washedthree times with 300 μl wash buffer (0.05% Tween 20 in PBS) and then 200μl reagent diluent (25% Tween 20 in PBS) was incubated in each well for1 h at room temperature. Diluted samples, standards (125, 250, 500,1000, 2000, 4000 and 8000 pg/ml A1AT) or blanks were then added to eachwell in duplicate and the plates were covered with a plate sealer andleft at room temperature for 2 h. At the end of the sample incubationstep, samples were removed and all wells washed as previously and 100 μldetection antibody (1:180 dilution from stock) was added to each welland incubated for a further 2 h at room temperature. Followingincubation with detection antibody, supernatant was removed and wellswere washed as previously and 100 μl streptavidin-HRP solution (1:200dilution from stock) was added to each well for 20 min in the dark.After which, 50 μl stop solution (2M H₂SO₄) was added and opticaldensity (OD) of each well was read at 450 nm with 570 nm blanksubtracted from each well using a microplate reader. A 4 parameterlogistic curve was constructed using GraphPad Prism 7 and A1ATconcentrations were determined in each sample by interpolation from astandard curve and multiplying by the appropriate dilution factor.

Results

The amount of human A1AT secreted from transfected HEK-EBNA cells intothe media was measured by ELISA. SAHA at 10 μM was used a positivecontrol for all in vitro A1AT secretion experiments.

The data in FIG. 3 show thatN,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide does notstimulate secretion of Siiyama A1AT at 1 or 10 μM, as measured by ELISA.In contrast, the positive control 10 μM SAHA stimulates an increase inSiiyama A1AT secretion.

Example 7: Activity ofN,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide in huZ Mice

The huZ mouse (also referred to as the PiZZ mouse) is a transgenic mousestrain that contains multiple copies of the Z variant of the human A1ATgene, developed by two separate groups Dycaico et al (Science (1988)242:1409-12) and Carlson et al (J. Clin Invest (1989) 83:1183-90). PiZZmice are on a C57Bl/6 background and express the human Z A1AT protein inliver tissue. The mice used in this study are from the progeny ofCarlson and colleagues (transgenic line Z11.03). HuZ mice have been usedas a tool to assess the effects of compounds on either increasing thecirculating levels of Z A1AT in plasma or the effects of compounds onthe accumulation of Z A1AT polymers in the liver and associated liverpathology.

Methods

HuZ mice (n=4/group; male or female) with basal human A1AT plasma levelsof between 200-600 μg/ml were treated with either vehicle orN,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide at 5, 15 or 50mg/kg twice a day by oral gavage for 14 consecutive days. Mice hadaccess to food (standard mouse chow, SAFE diets) and water ad libitum.On study day 14, each mouse was dosed one hour prior to terminalprocedures. Blood was taken from each mouse from the tail vein onpre-dosing days −12, −7 and −5 and on dosing days 12, 13 and 14. Bloodwas collected into microvettes containing EDTA and plasma was preparedby centrifugation at 2700×g at 4° C. for 10 min. Plasma was aliquotedand stored at −80° C. for bioanalysis. Plasma samples from pre-dosingdays −12, −7 and −5 were used to determine mean basal levels of human ZA1AT for each mouse. Plasma samples collected the last three dosing daysof the study (days 12, 13 and 14) were used to determine the effect ofN,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide on Z A1ATsecretion by measuring human Z A1AT levels and comparing to basal levelsfor each mouse. Human Z A1AT levels in mouse plasma samples weremeasured by ELISA (Human Serpin A1/α₁-antitrypsin duo set ELISA, R& DSystems, DY1268) per manufacturer's instructions.

Briefly, a 96 well plate was coated with human A1AT capture antibodyovernight at room temperature (1:180 dilution from stock, 100 μl finalvolume/well). The capture antibody was then removed and wells washedthree times with 300 μl wash buffer (0.05% Tween 20 in PBS) and then 200μl reagent diluent (25% Tween 20 in PBS) was incubated in each well for1 h at room temperature. Diluted samples, standards (125, 250, 500,1000, 2000, 4000 and 8000 pg/ml A1AT) or blanks were then added to eachwell in duplicate and the plates were covered with a plate sealer andleft at room temperature for 2 h. At the end of the sample incubationstep, samples were removed and all wells washed as previously and 100 μldetection antibody (1:180 dilution from stock) was added to each welland incubated for a further 2 h at room temperature. Followingincubation with detection antibody, supernatant was removed and wellswere washed as previously and 100 μl streptavidin-HRP solution (1:200dilution from stock) was added to each well for 20 min in the dark.After which, 50 μl stop solution (2M H₂SO₄) was added and opticaldensity (OD) of each well was read at 450 nm with 570 nm blanksubtracted from each well using a microplate reader. A 4 parameterlogistic curve was constructed using GraphPad Prism 7 and A1ATconcentrations were determined in each sample by interpolation from astandard curve and multiplying by the appropriate dilution factor.

Results

The effect of N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamideon circulating levels of human Z A1AT was assessed in a huZ mouse model.Mice were treated for 14 consecutive days by oral gavage twice daily at5, 15 or 50 mg/kg.

The data in FIG. 4 show thatN,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide stimulatessecretion of human Z A1AT compared to baseline levels in huZ mice in adose dependent manner.

Example 8: Pharmacokinetics ofN,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide in Mice

N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide wasadministered to male C57BI/6 mice intravenously (2 mg/kg) or orally (10mg/kg) by gavage. Whole blood diluted with water was prepared from thesedosed animals over a time course up to 24 hours post dose to allow bloodconcentrations of drug to be measured by UPLC-MS/MS. The measured druglevels allowed calculation of the following parameters forN,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide:

Half-life in blood (t_(1/2))=1.9 h

Observed clearance=7.8 ml/min/kg

Volume of distribution (Vz)=1.3 l/kg

Oral C_(max)=9311 ng/ml

AUC_(all)=15551 ng·h/ml

AUC_(INF)=15564 ng·h/ml

Oral Bioavailability (F, AUC_(INF))=73%

Example 9: Pharmacokinetics of Compounds of the Invention in Rats

Compounds were administered to Sprague Dawley rats intravenously (2mg/kg) or orally (10 mg/kg) by gavage. Whole blood diluted with waterwas prepared from these dosed animals over a time course up to 24 hourspost dose to allow blood concentrations of drug to be measured byUPLC-MS/MS.

The measured compound levels after the administration ofN,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide allowedcalculation of the following parameters forN,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide:

Half-life in blood (t_(1/2))=55 min

Observed clearance=10.6 ml/min/kg

Volume of distribution (Vz)=0.84 l/kg

Oral C_(max)=5326 ng/ml

Oral AUC_(INF)=16465 ng·h/ml

Oral Bioavailability (F)=104%.

The plasma levels ofN-methyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide at various timepoints after a 10 mg/Kg oral dose ofN,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide were as shownin Table 1.

TABLE 1 Plasma levels of N-methyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide after oral dose of N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide Time Mean Plasma Level ofN-methyl-4-((6-oxopyrimidin- (min) 1(6H)-yl)methyl)benzamide (ng/ml) 5369 15 548 30 1047 60 1395 120 1465 240 528 480 224 1440 62

The plasma levels ofN-methyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide at various timepoints after a 2 mg/Kg iv dose ofN,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide were as shownin Table 2.

TABLE 2 Plasma levels of N-methyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide after iv dose of N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide Time Mean Plasma Level ofN-methyl-4-((6-oxopyrimidin- (min) 1(6H)-yl)methyl)benzamide (ng/ml) 251 6 78 10 115 15 102 30 257 60 321 240 90 480 13

The measured compound levels after the administration ofN-methyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide allowedcalculation of the following parameters forN-methyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide:

Half-life in blood (t_(1/2))=63 min

Observed clearance=10.6 ml/min/kg

Volume of distribution (Vz)=1.0 l/kg.

The measured compound levels after the administration ofN,N-bis(methyl-d₃)-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide allowedcalculation of the following parameters forN,N-bis(methyl-d₃)-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide:

Half-life in blood (t_(1/2))=52 min

Observed clearance=10.6 ml/min/kg

Volume of distribution (Vz)=0.72 l/kg.

Example 10: Mouse, Rat and Human Hepatocyte Stability of the Compoundsof the Invention

The intrinsic clearances (CL_(int)) and half-lives of the compounds ofthe invention were measured in a hepatocyte suspension of cryopreservedmale C57BL6 mouse hepatocytes, a hepatocyte suspension of cryopreservedrat hepatocytes or a mixed hepatocyte suspension of cryopreserved humanhepatocytes. Briefly, the compound was incubated with hepatocytesuspensions at 37° C. over a time course and the remaining compound ateach time point was assessed by mass spectrometry (UPLC-MS/MS). CL_(int)in mouse, rat and human hepatocytes was <3 μl/min/10⁶ cells for allcompounds. Half-life in mouse, rat and human hepatocytes was >460 minfor all compounds.

Example 11: Plasma Protein Binding of Compounds of the Invention

The extent to which compounds of the invention bound to plasma proteinssuch as albumin and alpha-1 acid glycoprotein within human, rat or mouseplasma was determined by rapid equilibrium dialysis. Compounds wereincubated at 5 μM for 4 hours at 37° C. Plasma protein binding forN,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide in mouseplasma was 37.3%, in rat plasma was 14.8% and in human plasma was 30.6%.

Example 12: Activity of the Compounds of the Invention AgainstCytochrome P450s

Using E. coli CYPEX membranes in combination with specific probesubstrates, the inhibition of individual CYPs by compounds of theinvention using the method described in Weaver et al., 2003, Drug MetabDispos 31:7, 955-966 was assessed. Results are shown in Table 3.

TABLE 3 In vitro CYP inhibition data for N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl) benzamide CYP Inhibition (IC50 - μM)1A2 2C9 2C19 2D6 3A4 >50 >50 >50 >50 >50

Example 13: Activity of Compounds of the Invention Against the HERGChannel

Compounds of the invention were tested for inhibition of cardiacpotassium (hERG) channels using the Patchliner automated patch clamp.6-Point concentration-response curves were generated using half-logserial dilutions from a maximum final test concentration of 100 μM. IC₅₀values were obtained from a 4-parameter logistic fit of theconcentration-response data.N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide was shown tohave an IC50>100 μM with 7% inhibition at 100 μM. Reference compoundvalues were consistent with those presented in the literature (Elkins etal., 2013 J. Pharm. Tox. Meth. 68:11-122).

Example 14: Activity of Compounds of the Invention Against a Panel ofEnzymes, Ion Channels and Receptors

Compounds of the invention were tested against the DiscoverX Safety47™panel. N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamideexhibited an exceptionally clean off target profile at 10 μM. No targetwas inhibited by more than 25% at this concentration.

Example 15: Aqueous Solubility of Compounds of the Invention

Compounds of the invention were shaken for 5 minutes with increasingamounts of water until a clear solution had formed. Using this methodthe solubility ofN,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide was greaterthan 100 mg/ml.

Example 16: Activity of Compounds of the Invention in a BacterialReverse Mutation Test

The purpose of the bacterial reverse mutation test is to assess themutagenic potential of the test item by its ability to revert thespecified bacterial strains from auxotrophic growth to prototrophy. Theassay detects point (gene) mutations at specific histidine or tryptophanloci, which may be induced by compounds that cause base pairsubstitutions or frameshift mutations in the genome of these organisms.Assays were performed in the presence and absence of an exogenousmammalian oxidative metabolising system (S-9 mix, a liverpost-mitochondrial fraction derived from the livers of Aroclor 1254treated rats), to mimic mammalian metabolism. The test methodology wasbased on established procedures for bacterial mutagenicity testing.

N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide was tested upto the guideline regulatory maximum dose level of 5000 μg/plate in allstrains in the presence and absence of S-p9 mix.

There was no evidence of toxicity and no precipitation.

There were no increases in revertant numbers, greater than the definedfold-increases (to twice the relevant negative control value for TA98,TA100 or WP2 uvrA or three times the relevant negative control value forTA1535 or TA1537), in any strain at any dose level ofN,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide, in thepresence or absence of S-9 mix, under plate incorporation conditions.

N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide was notmutagenic under the conditions of this test.

Example 17: Activity of Compounds of the Invention in an In VitroMicronucleus Assay

The purpose of the in vitro micronucleus assay is to assess the abilityof the test item to cause chromosome or spindle damage, or to interferewith the cell cycle, in ways which lead to the formation of micronucleiin the cytoplasm of TK6 cells. Chromosome defects are recognised asbeing the basis of a number of human genetic diseases. Micronuclei (MN)are the result of chromosome fragments or whole chromosomes remaining inthe cytoplasm and not being incorporated into the nuclei of newly formedcells. These arise from damage to chromosomes leading to acentricfragments which cannot attach to the mitotic spindle; damage to thespindle apparatus preventing a chromosome from attaching to it or otherinterference with cell division. Assays were performed in the presenceand absence of an exogenous mammalian oxidative metabolising system (S-9mix, a liver post-mitochondrial fraction derived from the livers ofAroclor 1254 treated rats), to mimic mammalian metabolism. Cells wereexposed to the test item for 3 hours and sampled 44 hours after thebeginning of treatment. As a number of chemicals have been reported asonly exerting positive effects following prolonged treatment, acontinuous treatment of 27 hours in the absence of S-9 mix was alsoincluded. This was equivalent to 1.5 to 2.0 times the average generationtime of cultured cells used in the assay. The test methodology was basedon established procedures for in vitro micronucleus testing.

N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide was tested upto the regulatory maximum dose level of 1 mM (260 mg/mL) in alltreatments. There was no precipitation and only slight cytotoxicity inthe 27 hour treatment only. The relative increase in cell count (RICC)at 260 mg/mL was 105% in the presence of S-9 mix with 3 hour treatment,98% in the absence of S-9 mix with 3 hour treatment and 74% in theabsence of S-9 mix with 27 hour treatment, when compared with theconcurrent negative control.

There were no statistically significant increases in percentage MN (%MN), either in the presence of S-9 mix with 3 hour treatment or in theabsence of S-9 mix with 3 hour or 27 hour treatment. There was asignificant trend in the absence of S-9 mix with 3 hour treatment, butas there were no statistically significant increases and all % MN countswere well within the 95% control limits of the HCD, this was consideredto be not relevant.

N,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide was neitherclastogenic nor aneugenic in the presence or absence of S 9 mix, underthe conditions of this test.

Example 18: Trypsin Inhibition by the Human Z A1AT Secreted in thePresence of Compounds of the Invention in the huZ Mouse

The aim of the experiment was to purify human Z A1AT from three HuZ micewhich had been treated with compoundN,N-dimethyl-4-((6-oxopyrimidin-1(6H)-yl)methyl)benzamide (22.1e, 24.1a,24.1b), then test the activity of the A1AT by reaction with increasingamounts of trypsin. Reaction of A1AT with the trypsin was analysed bywestern blot, probing with an anti-human A1AT antibody. A control plasmafrom a wild type mouse treated with the same compound (15.5b) was alsoincluded in the study.

Methods

Mice were treated withN,N-dimethyl-4-((6-oxopyrimidin-201(6H)-yl)methyl)benzamide at 200 mg/kgtwice a day by oral gavage for 10 consecutive days. Mice had access tofood (standard mouse chow, SAFE diets) and water ad libitum. On studyday 10, each mouse was dosed 3.5-4.5 hours prior to terminal procedures.Blood was taken from each mouse by cardiac puncture and was collectedinto tubes containing EDTA 25 and plasma was prepared by centrifugationat 2700×g at 4° C. for 10 min. Plasma was aliquoted and stored at −80°C. prior to analysis.

100 μl of mouse plasma was diluted to 300 μl with 20 mM Tris, 150 mMNaCl, 5 mM EDTA, pH 7.4 (reaction and wash buffer) prior topurification. Washed al-antitrypsin Select beads (150 μl) (GE LifeSciences) were then added to the plasma and mixed on a rocker for 30minutes. The beads were then separated from the plasma by centrifugationusing a spin column (Thermo Scientific). After washing the beads with5×100p reaction buffer, the A1AT was then eluted from the beads with3×100 μl 20 mM Tris, 150 mM NaCl, 0.6M MgCl₂, 5 mM EDTA, pH 7.4. TheA1AT was eluted directly into 300 ul reaction buffer to ensure that theA1AT did not aggregate. Residual A1AT in the plasma and plasmasupernatant was quantified using a human serpinA1 ELISA (Human SerpinA1/α₁-antitrypsin duo set ELISA, R& D Systems, DY1268) to confirmdepletion of human A1AT. The concentration of the eluted fractions werealso measured. The amount of eluted A1AT was determined by measuring byA280 using an extinction coefficient of 0.45.

The activity of the eluted A1AT was tested by assessing its ability toform a complex with trypsin. Reaction mixtures were made up as in Table4. Reactions were incubated for 5 minutes prior to the addition of 1 μl1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride. After afurther 10 minutes 21 μl SDS sample buffer was added and boiled for 5minutes. Samples were run on a 10% SDS gel and blotted ontonitrocellulose membrane. The membrane was then blocked with PBS 0.10%triton x-100, 5% skimmed milk powder, then probed with a rabbit antihuman α-1 antitrypsin antibody (AbD serotec 0640-5039) overnight at 4°C. After washing the membrane with PBS, 0.1% triton X-100, 0.1% skimmedmilk, the blots were probed with anti-rabbit IgG HRP conjugate. Afterfurther washes of the membrane the blots were developed usingSuperSignal West pico PLUS chemiluminescent substrate (ThermoScientific). The result is shown in FIGS. 7A and B.

TABLE 4 Experimental conditions for various treatments of isolated ZA1AT Approx. A1AT Trypsin Gel Molar Ratio (100 μg/ml, (86 μg/ml, lane(A1AT:trypsin) 185 μM) 370 μM) Buffer 1 1:0 10 μl 0 μl 10 μl 2 4:1 10 μl1.25 μl 8.75 μl 3 2:1 10 μl 2.5 μl 7.5 μl 4 1:1 10 μl 5 μl 5 μl 5  1:1.25 10 μl 6.25 μl 3.75 μl 6  1:1.5 10 μl 7.5 μl 2.5 μl 7 1:2 10 μl10 μl 0 μl 8 Trypsin 0 μl 5 μl 15 μl Control

Results

The data in FIGS. 7A and B show that no human A1AT could be detected inthe plasma from the wild type control mouse (15.5b—see FIG. 7B). Human ZA1AT was detected in the three other samples from huZ mice (see FIGS. 7Aand B). On reaction with increasing amounts of trypsin, all samplesdemonstrated complete reaction with trypsin as indicated by the loss ofthe native band (labelled “NA”) and generation of complex (“C”) orcleaved bands (“CA”), indicating that the Z A1AT in the eluted fractionis native and fully active.

1-13. (canceled)
 14. A method of inducing Z A1AT secretion comprisingadministering a compound represented by the structure of:

or a pharmaceutically acceptable salt of any one thereof.
 15. The methodof claim 14, wherein the compound is represented by:

or a pharmaceutically acceptable salt thereof.
 16. The method of claim14, wherein the compound is represented by:

or a pharmaceutically acceptable salt thereof.
 17. The method of claim14, wherein the compound is represented by:

or a pharmaceutically acceptable salt thereof.
 18. The method of claim14, wherein the administering to the subject in need thereof isconducted orally.
 19. A method of treating α₁-antitrypsin deficiency(AATD) comprising administering a compound represented by the structureof:

or a pharmaceutically acceptable salt of any one thereof.
 20. The methodof claim 19, wherein the compound is represented by:

or a pharmaceutically acceptable salt thereof.
 21. The method of claim19, wherein the compound is represented by:

or a pharmaceutically acceptable salt thereof.
 22. The method of claim19, wherein the compound is represented by:

or a pharmaceutically acceptable salt thereof.
 23. The method of claim19, wherein the administering to the subject in need thereof isconducted orally.